We are having trouble with our iPSCs generating colonies after thaw. How long should we wait for colonies to form and do you think it is a problem with our cryopreservation protocol?

Answer

Cryogenic preservation of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas:

1. Good cell condition. iPSC should be fed daily before cryopreservation to get the healthier condition. Cells should be frozen after being passaged for 2-4 days. Overgrowth might make poorly viability after thawing. Before the cryopreservation, cell clumps should be dissolved. The cryoprotectant may be hard to penetrate the cell cluster, which results in only a small part of cells surviving after thawing. Properly handle and gently harvest the cultures. When collecting iPSC, we recommend centrifuging at 200 -300 X g for 2min, and operating pipettors gently. 1-2 x 10⁶ cells/ml is the typical density of cryopreservation. Too high density might reduce the cell viability.
2. Correct use of the cryoprotective agent. The most common cryoprotectant is DMSO. The final concentration of around 10% is most often used. For iPSC cryopreservation, some researchers also tend to add FBS or Ficoll to the freezing media. There are also some commercial products available in the market. To gain high recovery efficiency, researchers should use fresh medium materials. The cryoprotectant mix should be prepared on the day of the experiment.
3. A controlled rate of freezing. The ideal cooling rate for cells is -1°C per minute. The best way to control cooling rates is by using electronic programmable freezing units. To control the cost, researchers can use the Corning® CoolCell™. When cells are stored into Corning cryogenic vials, the vials can be inserted into a room temperature CoolCell container. Place the CoolCell container upright into a -80°C freezer or dry ice locker.
4. Storage under proper cryogenic conditions. Liquid nitrogen freezers permit storage in the vapor phase above the liquid at a temperature between -140°C and -180°C. Using vapor phase storage greatly reduces the possibility of leaky vials or ampules exploding during removal.

When thawing the iPSC:

1. Cells should be thawed rapidly by placing the cryovials in a water bath set at 37°C.
2. Use pipettor to transfer the cell suspension into 10 X volume of medium, drop by drop. The operation should be gentle and slow.
3. The seeding density range for each 35mm well (6-well plate) is between 2×10⁵ – 1×10⁶ viable cells. Cells might attach to the Corning Matrigel® coated plate in 30 minutes after thawing. 70-80% confluence may be observed after 24-48 hours of plating.